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Release of recombinant human interleukin-2 from dextran-based hydrogels
Journal of Controlled Release 78 (2002) 1–13 www.elsevier.com / locate / jconrel
Release of recombinant human interleukin-2 from dextran-based hydrogels ´ a , C.J. de Groot b , W. Jiskoot a , W. den Otter b , W.E. Hennink a , * J.A. Cadee a
Faculty of Pharmacy, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences ( UIPS), Utrecht University, P.O. Box 80 082, 3508 TB Utrecht, The Netherlands b Faculty of Veterinary Science, Department of Biochemistry, Cell Biology and Histology, Utrecht University, P.O. Box 80 176, 3508 TD Utrecht, The Netherlands Received 20 February 2001; accepted 28 June 2001
Abstract In this study, the release of recombinant human interleukin-2 (rhIL-2) from methacrylated dextran (dex-MA) and (lactate-)hydroxyethyl methacrylated dextran (dex-(lactate-)HEMA) hydrogels with varying crosslink density was investigated. Hydrogels derived from dex-MA are stable under physiological conditions (pH 7 and 378C), whereas dex-HEMA and dex-lactate-HEMA hydrogels degrade due to the presence of hydrolytically sensitive esters in the crosslinks of the gels. The protein release profiles both the non-degradable and degradable dextran-based hydrogels showed that with increasing crosslink density of the gel, the release of rhIL-2 decreases. From dex-MA hydrogels with an initial water content above 70%, the rhIL-2 release followed Fickian diffusion, whereas from gels with an initial water content of 70% or lower the protein was fully entrapped in the hydrogel meshes. In contrast with non-degradable dex-MA hydrogels, degradable dex-lactate-HEMA gels with comparable network characteristics (degree of methacrylate substitution and initial water content) showed an almost zero-order, degradation controlled release of rhIL-2 in a time period of 5–15 days. This paper demonstrates that the release of rhIL-2 from non-degradable dex-MA and degradable dex-lactate-HEMA gels can be modulated by the crosslink density and / or the degradation characteristics of the hydrogel. Importantly, rhIL-2 was mainly released as monomer from the hydrogels and with good retention of its biological activity. 2002 Elsevier Science B.V. All rights reserved. Keywords: Interleukin-2; Lysozyme; Biodegradable hydrogel; Protein release, Dextran
1. Introduction Interleukin-2 (IL-2) is a cytokine, which is produced by activated helper T lymphocytes and is an important mediator of the immune response. Because *Corresponding author. Tel.: 131-30-253-6964; fax: 131-30251-7839. E-mail address: [email protected] (W.E. Hennink).
of this biological function, recombinant human IL-2 (rhIL-2) has been tested in clinical trials for treatment of tumors [1,2]. rhIL-2 is a relatively hydrophobic protein with limited aqueous solubility [3]. The addition of stabilizers like mannitol, Tween-20 and / or heparin [3,4] or sodium dodecyl sulfate (SDS) [4,5] to the formulation increases the solubility of rhIL-2 and prevents aggregation of the protein as well as loss of its biological activity. Since
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00483-7
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the protein has a short half-life, frequent administration of rhIL-2 is required to obtain a therapeutic effect, which can result in severe toxic side effects [6]. In order to circumvent these problems, drug delivery systems, like liposomes [7,8] and polymer matrices [9–13], have been investigated to increase the therapeutic efficacy and minimize the side effects of rhIL-2. However, rhIL-2 was released from these drug delivery systems in a time period of days with a large initial burst. Furthermore, for the preparation of polymeric microparticles like poly(lactic-co-glycolic acid) organic solvents are used that are known to affect protein stability [14–16]. As protein delivery system, hydrogels are gaining more interest. These gels consist of hydrophilic polymeric networks, which can absorb substantial amounts of water. Moreover, no organic solvents are used for their preparation. Owing to their high water contents, they show a good compatibility with proteins and living tissue [17–19]. In this study, macroscopic biocompatible dextranbased hydrogels [20,21] were investigated for the controlled release of rhIL-2. These hydrogels were obtained by radical polymerization of dextran derivatized with methacrylate groups (dex-MA), hydroxyethyl methacrylate groups (dex-HEMA) or lactatehydroxyethyl methacrylate groups (dex-lactateHEMA) in aqueous solution. Scheme 1 gives the chemical structures of the different polymerizable dextran derivatives. Hydrogels derived from dex-MA are stable under physiological conditions (pH 7 and 378C), whereas the dex-HEMA and dex-lactateHEMA hydrogels degrade because of the presence of
Scheme 1.
hydrolyzable esters in the crosslinks [20]. The hydrogels based on dex-lactate-HEMA degraded faster than gels based on dex-HEMA, most likely because the dex-lactate-HEMA gels have more hydrolytically sensitive groups in the crosslinks than the dexHEMA gels [20]. The release of rhIL-2 from these hydrogels was studied as a function of the crosslink density, i.e. initial water content of the gel and degree of methacrylate substitution (defined as the number of methacryloyl groups per 100 glucose units). The rhIL-2 release profiles were compared with lysozyme release profiles, because the molecular weight of lysozyme is about the same as that of rhIL-2. In brief, the aim of this study was to evaluate the release of rhIL-2 from macroscopic, degradable dexlactate-HEMA hydrogels to gain insight into the mechanism of release: diffusion and / or degradation controlled release. The release of rhIL-2 from nondegradable dex-MA hydrogels was studied as well, because the release of a protein from these gels is by diffusion and depends on the crosslink density of the gels [22]. Special attention was given to the structural integrity of rhIL-2 as well as its biological activity after being released.
2. Materials and methods
2.1. Materials Hydroxyethyl methacrylate (HEMA, 2-hydroxyethyl methylpropenoate, 95% by GC), hydroquinone monomethyl ether (.98% by HPLC), dextran (from Leuconostoc mesenteroides, T40), glycidyl methacrylate (GMA, (6)-2,3-epoxypropyl methylpropenoate, 95% by GC), N,N,N9,N9-tetramethylethylene diamine (TEMED), sodium perchlorate monohydrate and dimethyl sulfoxide (DMSO, ,0.01% water) were obtained from Fluka Chemie, AG, Buchs, Switzerland. L-lactide ((3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione, .99.5%) was purchased from Purac Biochem BV (Gorinchem, The Netherlands) and used without pretreatment. Stannous octoate (stannous (II) bis (2-ethylhexanoate), SnOct 2 , 95%) from Sigma Chemical Co., St. Louis, MO, USA was used as received. Toluene, 4-(N,Ndimethylamino) pyridine (DMAP, 99%) and N,N9-
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
carbonyl diimidazole (CDI, 98%) were purchased from Acros Chimica (Geel, Belgium). Tetrahydrofuran (THF) was distilled from LiAlH 4 immediately before use. Acetonitrile (HPLC, gradient grade) was obtained from Biosolve LTD (Valkenswaard, The Netherlands). Dialysis tubes (cellulose, molecular weight cut off (based on proteins)512–14 kDa) were purchased from Medicell International Ltd., London, UK. Potassium peroxodisulfate (KPS), sodium azide (NaN 3 ), perchloric acid (70%, p.a.) and the phosphate buffer components were obtained from Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) was purchased from OPG Farma, The Netherlands. Sodium- 125 iodide (Na 125 I) was from Amersham. Lysozyme (from chicken egg white) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bradford protein assay (Bio-Rad protein assay concentrate) and silver stain plus kit were obtained from Bio-Rad Laboratories (Veenendaal, The Netherlands). RPMI-1640 medium was purchased from Life Technologies (Breda, The Netherlands) and the cell proliferation kit II (sodium 39-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid; XTT) was used from Roche Diagnostics Nederland BV, Almere, The Netherlands.
2.2. Interleukin-2 Recombinant human interleukin-2 (Proleukin; 1.2 mg rhIL-2 per vial) was kindly provided by Chiron B.V. (Amsterdam, The Netherlands). rhIL-2 is a protein with a molecular weight of 15.3 kDa [23]. It is produced by recombinant DNA technology using an Escherichia coli strain, which contains a genetically engineered modification of the human IL-2 gene. This modified rhIL-2 differs from native human IL-2: the protein is not glycosylated, it has no N-terminal alanine and the serine at position 125 is substituted for cysteine. The biological activities of rhIL-2 and native human IL-2 are similar [23]. When the white lyophilized powder is reconstituted with 1.2 ml water each vial contains per ml solution: 1 mg (18310 6 IU) rhIL-2, 50 mg mannitol (5% w / v), and 0.2 mg (0.02% w / v) SDS, buffered with sodium phosphates to a pH of 7.5 (range 7.2–7.8). This solution is further described as ‘rhIL-2 stock solution’.
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2.3. Methods 2.3.1. Radioiodination of rhIL-2 rhIL-2 was labeled with 125 I using the Iodo-gen method [24]. Briefly, a 50 ml aliquot (containing 25 mg) of Iodo-gen solution in dichloromethane was dispensed into a glass tube. The solvent was removed under a stream of nitrogen and 10 ml of 0.5 M Tris–HCl buffer (pH 7.4), 60 ml of 5% glucose containing 0.01% SDS, 15 ml of ‘rhIL-2 stock solution’ (1 mg / ml) and 8–10 ml of Na 125 I (.1 mCi) were added subsequently. After mixing, the mixture was incubated for 10 min at room temperature. The separation of labeled rhIL-2 from unreacted Na 125 I was performed by gel filtration using a Sephadex G-25 PD 10 column. As eluent 5% glucose containing 0.01% SDS was used. The labeling efficiency was more than 95% and the specific activity was 18 mCi / mg rhIL-2. The half-life of 125 I is 60 days [25]. 2.3.2. Stability of rhIL-2 in various buffers The stock solution of rhIL-2 (1 mg / ml) was diluted to 100 mg / ml with 0.9% NaCl; with 10 mM phosphate buffer (PB) or 10 mM phosphate buffered saline (PBS), 10 mM PB containing 0.01% SDS and 5% glucose, PB (10 mM and 100 mM) containing 0.1% SDS or 20 mM HEPES with or without 0.8% NaCl. The pH of the buffers was 7.2 and the total volume was 1 ml. Firstly, the stability of rhIL-2 in these buffers was tested for a short incubation period (15 min, room temperature). Secondly, the stability of rhIL-2 was studied over a longer time period (30 days) using the buffer (100 mM PB containing 0.1% SDS) in which rhIL-2 was recovered completely after 15 min of incubation. Now, 0.02% sodium azide (NaN 3 ) was added to avoid bacterial growth. The protein (36 mg / ml) was incubated in 15 ml buffer under physiological conditions (pH 7.2 and 378C) and samples were taken periodically. The samples were centrifuged (Eppendorf, 13 0003g) and 0.5 ml of the supernatant was removed and assayed for the presence of rhIL-2 by RP-HPLC (see Section 2.3.8). The same experiments were performed using 125 IrhIL-2. Briefly, 10 ml (0.9 mCi) of 125 I-rhIL-2 was added to rhIL-2 with a final concentration of 100
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mg / ml. The samples were centrifuged (Eppendorf, 13 0003g) and the number of counts in 0.5 ml of the supernatant and in the remaining resuspended fluid (0.5 ml) was determined using a gamma counter 1470 Wizard (EG&G Wallac, Turku, Finland).
2.3.3. Preparation of dextran hydrogels The polymers, dex-MA and dex-lactate-HEMA, were prepared from dextran T40 as previously described [26–28]. In brief: dex-MA was synthesized by the coupling of glycidyl methacrylate to dextran dissolved in DMSO using 4-(N,N-dimethylamino)pyridine as catalyst. For the synthesis of Dexlactate-HEMA, first L-lactide was grafted onto HEMA using stannous octoate as catalyst, yielding HEMA-lactate. After activation of the terminal hydroxyl group of HEMA-lactate with N,N9-carbonyldiimidazole, the resulting HEMA-lactate-CI was coupled to dextran in DMSO using 4-(N,N-dimethylamino)pyridine as catalyst to yield dex-lactateHEMA. Hydrogels were obtained by radical polymerization of dex-MA or dex-lactate-HEMA in aqueous solution according to the following general procedure [20]. For a hydrogel with an initial water content of 80% (w / w), 400 mg of methacrylated dextran was dissolved in 1460 ml phosphate buffer (PB; 100 mM, pH 7.2), in a 2-ml Eppendorf cup. To the dextran solution, 100 ml of KPS (20 mg / ml) in 100 mM phosphate buffer (PB, pH 7.2) was added and mixed well. Subsequently, 40 ml of a 20% (v / v) TEMED solution in PB (adjusted with 4 N HCl to pH 7) was added. After mixing, the resulting solution was allowed to polymerize for 1 h at room temperature. 2.3.4. Degradation of hydrogels After polymerization, the dex-lactate-HEMA hydrogels were removed from the cups, cut into a cylindrical shape (length approximately 2.5 cm, radius 0.45 cm), and accurately weighed (W0 ). The hydrogels were transferred into glass vials containing 15 ml of phosphate buffered saline (100 mM PB, 0.9% NaCl, 0.02% NaN 3 , pH 7.2) or phosphate buffer (100 mM PB) containing 0.1% SDS and 0.02% NaN 3 , pH 7.2. After incubation in a water bath at 378C, the weight of the hydrogels was determined at regular time points, and used to calculate the swelling ratio. The swelling ratio is
defined as the ratio of the weight of the hydrogel at time t (Wt ) and the initial weight of the hydrogel (W0 ) [20].
2.3.5. Release of proteins from dextran hydrogels The protein-loaded dex-MA and dex-lactateHEMA hydrogels were prepared according to the procedure described in Section 2.3.3. For lysozyme loaded gels, 25 ml of a 100 mg / ml lysozyme solution in PB was added to the polymer solution, yielding a loading of 1.25 mg lysozyme / g gel. For rhIL-2 loaded hydrogels, 420 ml of a 1.3 mg / ml rhIL-2 solution was added to the polymer solution, yielding a loading of 0.27 mg rhIL-2 / g gel. The release of lysozyme from the gels was determined after incubation of the protein loaded gel at 378C in vials containing 15 ml 100 mM PB containing 0.9% NaCl and 0.02% NaN 3 (PBS), pH 7.2. The rhIL-2 loaded gels were incubated in 15 ml of 100 mM PB containing 0.1% SDS and 0.02% NaN 3 , pH 7.2. The vials were gently shaken, and 1 ml samples were taken periodically and replaced by fresh buffer. The lysozyme concentration was measured with the Bio-Rad protein assay [29]. The rhIL-2 concentration was determined by RP–HPLC. 2.3.6. Release of 125 I-rhIL-2 from dextran hydrogels 125 I-rhIL-2 loaded hydrogels were prepared as described above. Before polymerization the polymer solution contained per ml 0.8 mCi 125 I-rhIL-2 and 0.27 mg non-labeled rhIL-2. After 1 h polymerization at room temperature, the gel was incubated in 15 ml of 100 mM PB10.1% SDS10.02% NaN 3 , pH 7.2. Periodically, 1 ml samples were taken and replaced by fresh buffer. The number of counts was measured with a gamma counter 1470 Wizard. The percentage of free label ( 125 I) in the release samples was determined by ITLCESG Instant Thin Layer Chromatography (silica gel impregnated glass fiber sheets; Gelman Sciences, Ann Arbor, MI, USA). As eluent 0.15 M sodium citrate pH 5.5 was used. The protein-loaded gels which were still present after 35 days, were transferred into a glass tube and the amount of remaining rhIL-2 in the gel was measured by using a gamma counter 1470 Wizard.
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
2.3.7. Adsorption of rhIL-2 onto dextran-based hydrogels To investigate the possible adsorption of rhIL-2 onto dextran-based hydrogels, gels with initial water content of 70% and DS 7-9 were incubated in an rhIL-2 solution with varying protein concentration. Solutions with 7 and 36 mg / ml rhIL-2 in 100 mM PB containing 0.1% SDS and 0.02% NaN 3 with or without 125 I-rhIL-2 (0.8 mCi) were used. Periodically, samples were taken and analyzed with RP– HPLC or a gamma-counter 1470 Wizard. 2.3.8. High-performance liquid chromatography rhIL-2 samples were analyzed by reversed phase– high-performance liquid chromatography (RP– HPLC) [30]. An LC Module I system (WatersE) ˚ with an analytical column (Jupiter, 5 mm C4 300 A, 15034.6 mm including a SecurityGuardE guard cartridge system with Widepore C4 (433 mm)) was used. All rhIL-2 samples were centrifuged for 5 min (13 0003g) and 100 ml of the supernatant was injected onto the column. A linear gradient was run from 40% A (water–acetonitrile 95:5; w / w; 100 mM sodium perchlorate (NaClO 4 ); 10 mM perchloric acid (HClO 4 )); 60% B (water–acetonitrile 5:95; w / w; 100 mM NaClO 4 ; 10 mM HClO 4 ) to 100% B in 10 min. The flow-rate was 1.0 ml / min and the column oven was set at 308C. UV detection at a wavelength of 205 nm was applied. Peak areas were determined with Millennium 2010 V. 2.15 software (Waters Associates Inc.). The total amount of methionine oxidized [31] and native rhIL-2 was calculated by using an rhIL-2 calibration curve over the range of 0.1–5 mg IL-2. The relative standard deviation for the intra-day precision was less than 3% and the inter-day standard deviation was 7%, which is within the range of the methodical error [32,33]. 2.3.9. Gel electrophoresis SDS–PAGE based on the method of Laemmli [34] was performed under denaturing (non-reducing and reducing) and native conditions. The Bio-Rad MiniProtean system was used with a 5% stacking gel and a 12.5% separating gel (thickness of 0.75 mm), both containing 0.4% SDS (denaturing conditions) or 0.1% SDS (native conditions). The rhIL-2 concen-
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tration was in the range of 3–25 mg / ml. For denaturing conditions (non-reducing), sample buffer (60 mM Tris–HCl, pH 6.8; 25% (v / v) glycerol; 2% (v / v) SDS; 0.1% (w / v) bromophenol blue) was added to the samples in 1:4 (v / v) buffer to sample ratio and subsequently heated at 1008C for 5 min. For reducing conditions the sample buffer also contained b-mercaptoethanol (5% (v / v)). Native gel electrophoresis (non-reducing, non-boiled) was also performed. The sample buffer (60 mM Tris–HCl, pH 6.8; 25% (v / v) glycerol; 0.5% (v / v) SDS; 0.1% (w / v) bromophenol blue) was added to the samples in 1:4 (v / v) buffer to sample ratio. After loading the samples (24 ml sample per well), the gel was run for approximately 2 h at a voltage of 100 V with an electrophoresis buffer (0.3% (w / v) Tris; 1.4% (w / v) glycine; 0.1% (v / v) SDS). After running, the protein bands were visualized by silver nitrate staining. The marker contained lysozyme (Sigma, chicken egg white; Mw 15 kDa), ovalbumin (Sigma, chicken egg; Mw 40 kDa) and BSA monomer (ICN, Mw 66 kDa) in 100 mM PB containing 0.1% SDS. The concentration was 20 mg / ml for each protein. As control, rhIL-2 containing 6% of dimer was used (kindly provided by Chiron BV, Amsterdam, The Netherlands).
2.3.10. rhIL-2 bioassay The biological activity of rhIL-2 was measured by using an rhIL-2 dependent murine tumor-specific cytotoxic T cell line (CTLL-2) [35]. Samples of rhIL-2 released at different time points from dex-MA DS 9 and dex-lactate-HEMA DS 7 hydrogels with different initial water contents were tested. The rhIL2 concentration was determined by RP–HPLC. As control, rhIL-2 was incubated in the release buffer at 378C. Cells were cultured in RPMI-1640 medium supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin and 10% fetal calf serum in a humidified atmosphere of 5% CO 2 in air at 378C. The samples were diluted in steps of 1 in 10 in a range of 30 ng / ml to 0.03 ng / ml in a 96-wells plate (100 ml per well; in triplicate). To each well 50 ml of CTLL-2 cell suspension (10 000 cells) was added and the plate was incubated for 2 days in a humidified atmosphere of 5% CO 2 in air at 378C. Then, 50 ml of XTT solution was added to each well
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and after 4–12 h the proliferation was measured by using a Cell Proliferation Kit II.
3. Results and discussion
3.1. Stability of rhIL-2 As pointed out in the introduction, the rhIL-2 formulation upon reconstitution and dilution is critical since the protein has limited aqueous solubility [3,5,24]. To examine in which aqueous solution rhIL-2 is stable, the protein was incubated in various media. The recovery of rhIL-2 after 15 min incubation at room temperature was less than 80% when the protein was diluted in 0.9% saline, PB with or without saline, PB containing 0.01% SDS and 5% glucose, or in HEPES buffer with or without saline. The addition of a detergent, 0.1% SDS, to the PB without saline resulted in complete solubilization of rhIL-2 (protein recovery .95%). rhIL-2 also showed a good long term stability in this buffer solution: in a time period of 30 days only a slight decrease of approximately 10% in rhIL-2 recovery was detected. Importantly, rhIL-2 incubated in this buffer showed no detectable loss in biological activity (14 days, 378C). Moreover, in line with this observation aggregates of rhIL-2 were not detected as determined with native gel electrophoresis (Fig. 1A; lane 9). Therefore, the release studies of rhIL-2 from hydrogels were performed in 100 mM PB containing 0.1% SDS and 0.02% NaN 3 , pH 7.2.
3.2. Degradation of dex-lactate-HEMA hydrogels From the stability study of rhIL-2 in different aqueous media, it was shown that rhIL-2 was stable in 100 mM PB containing 0.1% SDS and not in PBS. Since the degradation of dextran-based gels was only examined in PBS [20], these gels were incubated in PB containing 0.1% SDS to investigate whether SDS has an effect on the degradation process. Fig. 2 shows a representative example of the swelling behavior of a dex-lactate-HEMA gel with initial water content of 80% and DS of 7 in PBS and in PB containing 0.1% SDS. The hydrogel showed in both buffers a progressive swelling, which is caused by hydrolysis of the lactate and / or carbonate esters
Fig. 1. Native gel electrophoresis (A) and SDS–PAGE analysis under reducing conditions (B) of rhIL-2 release samples of dexMA DS 9 and dex-lactate-HEMA DS 7 gels both with initial water content of 90%. Lane 1: Molecular weight marker (BSA, ovalbumin and lysozyme); lane 2: rhIL-2 standard containing 6% dimer; lanes 3–5: rhIL-2 released from dex-MA gel incubated in 100 mM PB10.1% SDS after 1, 3 and 7 days, respectively; lanes 6–8: rhIL-2 release from dex-lactate-HEMA gel incubated in 100 mM PB10.1% SDS after 1, 3 and 7 days, respectively. As control, rhIL-2 was incubated for 17 days in 100 mM PB containing 0.1% SDS at 378C (1A; lane 9).
present in the crosslinks, followed by a dissolution phase [20]. The time in which the gel is completely degraded is defined as the degradation time of the gel. The degradation time of the dex-lactate-HEMA hydrogel was longer when the buffer contained SDS (Fig. 2 and Table 1). This can be ascribed to the interaction of SDS with the hydrophobic lactate units present in the hydrogel. A likely explanation is that shielding diminishes the accessibility of water molecules to hydrolyze the lactate ester bonds, resulting in a slower degradation of the dex-lactate-HEMA
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Fig. 2. Swelling behavior of dex-lactate-HEMA hydrogels with DS 7 and an initial water content of 80% in 100 mM PBS (h) or in 100 mM PB containing 0.1% SDS, pH 7.2 (j), at 378C. The values are the average of two independent measurements.
hydrogel and an increased degradation time in an SDS-containing buffer.
Fig. 3. Representative HPLC chromatogram of rhIL-2 standard (A), rhIL-2 released from an 80% dex-MA DS 9 gel after 5 days (B) and rhIL-2 released from a 90% dex-lactate-HEMA DS 7 gel after 5 days (C; peaks with retention times of 4–6 min correspond to degradation products of the dex-lactate-HEMA gel).
3.3. Protein release from dextran-based hydrogels The release of rhIL-2 and lysozyme from nondegradable dex-MA hydrogels and from degradable dex-lactate-HEMA gels with different crosslink densities was studied. In contrast to the rhIL-2 standard (Fig. 3A), HPLC chromatograms of rhIL-2 released from the hydrogels showed two peaks with a retention time of 8 and 9.5 min (Fig. 3B,C). The peak with the largest retention time (9.5 min) contains native as well as methionine oxidized forms (20– 33%) of rhIL-2. The peak with the shortest retention time (8 min) contains rhIL-2 in which all methionine Table 1 The degradation time of dex-lactate-HEMA DS 7 hydrogels with varying initial water content (w / w) in 100 mM PBS, pH 7.2 and 100 mM PB containing 0.1% SDS, pH 7.2 Initial water content (%) 90 80 70 60
Degradation time (days) PBS
PB10.1% SDS
5 9 18 23
7 15 31 36
residues at position 103 are oxidized and also oxidized methionine residues at the other positions [31,36]. Fig. 4 shows the release of rhIL-2 (A) and lysozyme (B) from dex-MA hydrogels with varying initial water content. As demonstrated previously [22], gels with a DS around 10 are dimensionally stable (do not show additional water uptake once incubated in an aqueous solution). This means that the equilibrium water content is the same as the water content of the gels after preparation. Fig. 4 shows that, as expected, with decreasing water content of the hydrogel, the release rate as well as the total release of both proteins decrease. Similar release profiles of rhIL-2 from dex-MA hydrogels were obtained when 125 I labeled rhIL-2 was used (not shown). Both HPLC and radioactivity measurements showed that rhIL-2 was not quantitatively released from the investigated gels in the time frame studied. The non-released part was still entrapped in the hydrogel: using 125 I-rhIL-2, it was shown that 6 and 80% of the initial amount of protein was still present after 35 days in gels with initial water
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Fig. 4. Release of rhIL-2 (A) and lysozyme (B) from dex-MA hydrogels with DS 9 and an initial water content of 90% (j), 80% (♦), 75% (m), 70% (d), 60% (.) and 50% (w) in 100 mM PB pH 7.2, with (rhIL-2) or without 0.1% SDS, at 378C. Duplicate release curves varied less than 10% as indicated by the error bars. Inserts show the initial proteins release of rhIL-2 from these hydrogels versus the square root of time.
contents of 90 and 70%, respectively. The total amount of released rhIL-2 and still entrapped rhIL-2 in the gels resulted in a recovery of more than 90%. As shown in Fig. 4, substantial amounts of rhIL-2 and lysozyme were released from dex-MA hydrogels
with initial water content of 75–90% and 50–90%, respectively. The initial release of both proteins from these hydrogels is proportional to the square root of time as depicted in the insert of Fig. 4A and B. This indicates that the pore sizes of the hydrogels are
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larger than the hydrodynamic radius of the protein [22] and that the release followed Fickian diffusion [37,38]. Fig. 4 shows that for rhIL-2 a slower release was observed than for lysozyme from gels with the same network characteristics. Moreover, rhIL-2 was marginally released (less than 10% in 25 days) from dex-MA gels with an initial water content of 70% or lower (Fig. 4A), whereas a significant amount of lysozyme was released from gels with the same composition (Fig. 4B). This suggests that in gels with an initial water content of #70% rhIL-2 is almost fully entrapped in the meshes of the gels, whereas (part of) lysozyme is still able to diffuse through the network. The entrapment of rhIL-2 is unexpected, since the molecular weight of the monomer of rhIL-2 is almost the same as that of lysozyme. Possible explanations might be interaction of rhIL-2 with the hydrogel matrix, formation of aggregates or interference by SDS. Native gel electrophoresis showed that some dimers of rhIL-2 are released from a dex-MA gel with initial water content of 90% (Fig. 1A lanes 3–5). To investigate whether these dimers consist of disulfide-linked protein species, SDS–PAGE under non-reducing and reducing conditions was performed. As shown in Fig. 1B (lane 3–5), the dimers were still present under reducing conditions suggesting that the dimers are linked by covalent bonds other than disulfide bridges. However, the amount of rhIL-2 dimer released is approximately 5–10% based on rhIL standard containing 6% dimer (Fig. 1B; lane 2). Thus, the entrapment of rhIL-2 in dex-MA gels with initial water content of 70% or lower can not be fully explained by the formation of dimers. Possible adsorption of rhIL-2 to the hydrogel matrix was investigated by incubation of a dex-MA gel in an rhIL-2 solution. This resulted in no adsorption of rhIL-2 (RP–HPLC, 125 I-rhIL-2) to the gel, indicating that the protein has no interaction with the dex-MA hydrogel matrix. Since it is likely that SDS interacts with the crosslinks (polymerized methacrylate groups) in the gel, the matrix becomes negatively charged. On the other hand, SDS will also interact with rhIL-2, resulting in a larger hydrodynamic protein diameter and an overall negative charge. It can be expected that the mobility of the negatively charged SDS coated rhIL-2 molecules in such a
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matrix is restricted and might explain the observed entrapment of rhIL-2 in matrices with small pores [39]. The release of both rhIL-2 and lysozyme from degradable dex-lactate-HEMA and dex-HEMA hydrogels was also investigated. Fig. 5A and B show the release profiles of rhIL-2 and lysozyme, respectively, from dex-lactate-HEMA gels with varying initial water content. Since the degradation products
Fig. 5. (A) Release of rhIL-2 from dex-lactate-HEMA hydrogels with DS 7 and an initial water content of 90% (j), 80% (♦), 75% (m), 70% (d) and 60% (.) in 100 mM PB containing 0.1% SDS, pH 7.2, 378C. (B) Release of lysozyme release from dex-lactateHEMA hydrogels with DS 7 and an initial water content of 90% (j), 80% (♦), 70% (d) and 60% (.) in 100 mM PBS, pH 7.2, 378C. Insert shows the initial release of lysozyme from these hydrogels versus the square root of time.
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(among others oligomethacrylates) interfered with the protein assay [20] and most likely with the HPLC analysis as well, the release of both proteins from the degrading hydrogels was quantified until the matrix of the gels started to dissolve. As expected, by decreasing the initial water content of the gel, the protein release rate decreases. As observed for the non-degradable dex-MA gels, the release of lysozyme from the dex-lactate-HEMA gels showed a square root of time dependency (insert of Fig. 5B). This indicates that the pore sizes of these hydrogels are larger than the hydrodynamic radius of lysozyme and the lysozyme release from these hydrogels therefore followed Fickian diffusion. The release of rhIL-2 from a highly hydrated dex-lactate-HEMA gel (initial water content of 90%) showed a square root of time dependency as well (data not shown). In contrast to the marginal rhIL-2 release from nondegradable dex-MA gels with initial water content of 70% or lower (Fig. 4A), rhIL-2 was released from degradable dex-lactate-HEMA gels with similar crosslink densities (Fig. 5A). This can be ascribed to hydrolysis of the lactate esters present in the crosslinks, resulting in an increased mesh size of the gel in time. Now, the protein release is controlled by the swelling of the matrix [20]. An almost zero-order release of rhIL-2 in a time period of approximately 5–15 days was obtained from the dex-lactate-HEMA gels (initial water content ,90%). The observed zero-order release of rhIL-2 can be explained as described previously for the release of immunoglobulin G from gels with the same composition [20]. On the one hand the diffusion coefficient of the protein increases in time due to swelling of the matrix; on the other hand the release rate decreases due to increase in hydrogel size and decrease in concentration gradient. Obviously in the dex-lactate-HEMA hydrogels, these factors compensate each other, resulting in an almost zero-order release. To investigate the release of rhIL-2 from dexlactate-HEMA hydrogels over a longer time period, 125 I labeled rhIL-2 was encapsulated in these hydrogels. The release profiles established by measuring the radioactivity of the samples matched with those determined by RP–HPLC, especially for a hydrogel with a high initial water content (Fig. 6). Moreover, the recovery of 125 I-rhIL-2 after complete degra-
Fig. 6. Release of rhIL-2 loaded dex-lactate-HEMA hydrogels as measured by radioactivity of co-incorporated 125 I-rhIL-2 (closed symbols) or RP–HPLC (open symbols). The initial water content of the hydrogels was 90% (squares) and 70% (circles).
dation of the hydrogels was approximately 90% as shown in Fig. 6. As was true for the dex-MA hydrogels, rhIL-2 released from the dex-lactateHEMA gels consisted of mainly monomer of rhIL-2 and some covalently bonded dimers (5–10%; Fig. 1A1B lanes 6–8). The release of rhIL-2 from degradable dex-HEMA gels with fixed initial water content of 70% and different degree of substitutions, DS 8 and 14, was also studied (Fig. 7). 125 I-rhIL-2 was used since the degradation products of dex-HEMA hydrogel might interfere with HPLC analysis. The release of rhIL-2 is slower from a degradable dex-HEMA gel as compared with a dex-lactate-HEMA gel with comparable network characteristics (DS and initial water content; Fig. 7). These differences in release profile can be explained by the faster degradation of the dex-lactate-HEMA gel compared to dex-HEMA gels (1 and 3 months, respectively). Furthermore, by increasing the degree of substitution of the dex-HEMA gels, the rhIL-2 release rate decreases. It is obvious that increasing the DS at a fixed water content results in a network with a higher crosslink density. To dissolve the network more crosslinks have to be hydrolyzed resulting in a slower swelling, which is associated with a slower protein release.
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
11
polyphosphate pastes showed a loss of approximately 70% in biological activity [40]. Gel electrophoresis showed that rhIL-2 was mainly released as monomer from both non-degradable dex-MA and degradable dex-lactate-HEMA gels (Fig. 1A and B). Since the concentration of released rhIL-2 was too low for spectroscopic analysis like circular dichroism or fluorescence spectroscopy, the protein integrity was studied by measuring its biological activity. As shown in Table 2, rhIL-2 released from hydrogels has 50–70% of its original activity as evaluated by the CTLL-2 proliferation assay. The nature of the observed loss of the bioactivity is under present investigation. Fig. 7. Release of 125 I-rhIL-2 from dex-HEMA hydrogels with DS 8 (♦) and DS 14 (d) with initial water contents of 70%. As control, the release of rhIL-2 from a 70% dex-lactate-HEMA DS 7 (j) gel.
3.4. Biological activity Proteins that are encapsulated in release systems, can be damaged during the formulation, storage and release, which can result in loss of biological activity [16]. Egilmez et al. reported that half of the biological activity of rhIL-2 is lost during the encapsulation process of the protein in polylactic acid microspheres. This is most likely a result of protein denaturation, caused by the presence of organic solvents [13]. RhIL-2 released from biodegradable
4. Conclusions In this paper, we demonstrate that the release of rhIL-2 from dextran-based hydrogels can be tailored by the crosslink density and the type of ester group present in the crosslink. From non-degradable dexMA gels with an initial water content above 70% the rhIL-2 release was governed by diffusion, whereas in gels with an initial water content of 70% or lower the protein was entrapped. The rhIL-2 release from degradable dex-lactate-HEMA gels was determined by diffusion and / or hydrogel degradation. Importantly, the degradable rhIL-2 loaded hydrogels showed an almost zero-order release in a time period of approximately 5–15 days. rhIL-2 released from the
Table 2 Biological activity of rhIL-2 released from dex-MA and dex-lactate-HEMA gels with initial water content (w / w) of 70% and 90% at different time points Formulation
Water content (%)
Day
Biological activity (%)a
Dex-MA
90
Dex-lactate-HEMA
90
2 8 2 7 11
79 65 65 81 64
Dex-MA
70
Dex-lactate-HEMA
70
1 19 1 24
46 53 57 55
a
The biological activity is related to freshly reconstituted rhIL-2 in PB containing 0.1% SDS. The values are the mean of two measurements that deviated 5–10%.
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dextran hydrogels investigated was mainly in its monomeric form with a partial preservation of the biological activity (50–70%). rhIL-2 loaded dextranbased hydrogels are promising protein delivery systems for tumor immunotherapy.
[10]
[11]
Acknowledgements [12]
This investigation was supported by the Dutch Technology Foundation, research grant UFA 55.3931.
[13]
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Release of recombinant human interleukin-2 from dextran-based hydrogels ´ a , C.J. de Groot b , W. Jiskoot a , W. den Otter b , W.E. Hennink a , * J.A. Cadee a
Faculty of Pharmacy, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences ( UIPS), Utrecht University, P.O. Box 80 082, 3508 TB Utrecht, The Netherlands b Faculty of Veterinary Science, Department of Biochemistry, Cell Biology and Histology, Utrecht University, P.O. Box 80 176, 3508 TD Utrecht, The Netherlands Received 20 February 2001; accepted 28 June 2001
Abstract In this study, the release of recombinant human interleukin-2 (rhIL-2) from methacrylated dextran (dex-MA) and (lactate-)hydroxyethyl methacrylated dextran (dex-(lactate-)HEMA) hydrogels with varying crosslink density was investigated. Hydrogels derived from dex-MA are stable under physiological conditions (pH 7 and 378C), whereas dex-HEMA and dex-lactate-HEMA hydrogels degrade due to the presence of hydrolytically sensitive esters in the crosslinks of the gels. The protein release profiles both the non-degradable and degradable dextran-based hydrogels showed that with increasing crosslink density of the gel, the release of rhIL-2 decreases. From dex-MA hydrogels with an initial water content above 70%, the rhIL-2 release followed Fickian diffusion, whereas from gels with an initial water content of 70% or lower the protein was fully entrapped in the hydrogel meshes. In contrast with non-degradable dex-MA hydrogels, degradable dex-lactate-HEMA gels with comparable network characteristics (degree of methacrylate substitution and initial water content) showed an almost zero-order, degradation controlled release of rhIL-2 in a time period of 5–15 days. This paper demonstrates that the release of rhIL-2 from non-degradable dex-MA and degradable dex-lactate-HEMA gels can be modulated by the crosslink density and / or the degradation characteristics of the hydrogel. Importantly, rhIL-2 was mainly released as monomer from the hydrogels and with good retention of its biological activity. 2002 Elsevier Science B.V. All rights reserved. Keywords: Interleukin-2; Lysozyme; Biodegradable hydrogel; Protein release, Dextran
1. Introduction Interleukin-2 (IL-2) is a cytokine, which is produced by activated helper T lymphocytes and is an important mediator of the immune response. Because *Corresponding author. Tel.: 131-30-253-6964; fax: 131-30251-7839. E-mail address: [email protected] (W.E. Hennink).
of this biological function, recombinant human IL-2 (rhIL-2) has been tested in clinical trials for treatment of tumors [1,2]. rhIL-2 is a relatively hydrophobic protein with limited aqueous solubility [3]. The addition of stabilizers like mannitol, Tween-20 and / or heparin [3,4] or sodium dodecyl sulfate (SDS) [4,5] to the formulation increases the solubility of rhIL-2 and prevents aggregation of the protein as well as loss of its biological activity. Since
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00483-7
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the protein has a short half-life, frequent administration of rhIL-2 is required to obtain a therapeutic effect, which can result in severe toxic side effects [6]. In order to circumvent these problems, drug delivery systems, like liposomes [7,8] and polymer matrices [9–13], have been investigated to increase the therapeutic efficacy and minimize the side effects of rhIL-2. However, rhIL-2 was released from these drug delivery systems in a time period of days with a large initial burst. Furthermore, for the preparation of polymeric microparticles like poly(lactic-co-glycolic acid) organic solvents are used that are known to affect protein stability [14–16]. As protein delivery system, hydrogels are gaining more interest. These gels consist of hydrophilic polymeric networks, which can absorb substantial amounts of water. Moreover, no organic solvents are used for their preparation. Owing to their high water contents, they show a good compatibility with proteins and living tissue [17–19]. In this study, macroscopic biocompatible dextranbased hydrogels [20,21] were investigated for the controlled release of rhIL-2. These hydrogels were obtained by radical polymerization of dextran derivatized with methacrylate groups (dex-MA), hydroxyethyl methacrylate groups (dex-HEMA) or lactatehydroxyethyl methacrylate groups (dex-lactateHEMA) in aqueous solution. Scheme 1 gives the chemical structures of the different polymerizable dextran derivatives. Hydrogels derived from dex-MA are stable under physiological conditions (pH 7 and 378C), whereas the dex-HEMA and dex-lactateHEMA hydrogels degrade because of the presence of
Scheme 1.
hydrolyzable esters in the crosslinks [20]. The hydrogels based on dex-lactate-HEMA degraded faster than gels based on dex-HEMA, most likely because the dex-lactate-HEMA gels have more hydrolytically sensitive groups in the crosslinks than the dexHEMA gels [20]. The release of rhIL-2 from these hydrogels was studied as a function of the crosslink density, i.e. initial water content of the gel and degree of methacrylate substitution (defined as the number of methacryloyl groups per 100 glucose units). The rhIL-2 release profiles were compared with lysozyme release profiles, because the molecular weight of lysozyme is about the same as that of rhIL-2. In brief, the aim of this study was to evaluate the release of rhIL-2 from macroscopic, degradable dexlactate-HEMA hydrogels to gain insight into the mechanism of release: diffusion and / or degradation controlled release. The release of rhIL-2 from nondegradable dex-MA hydrogels was studied as well, because the release of a protein from these gels is by diffusion and depends on the crosslink density of the gels [22]. Special attention was given to the structural integrity of rhIL-2 as well as its biological activity after being released.
2. Materials and methods
2.1. Materials Hydroxyethyl methacrylate (HEMA, 2-hydroxyethyl methylpropenoate, 95% by GC), hydroquinone monomethyl ether (.98% by HPLC), dextran (from Leuconostoc mesenteroides, T40), glycidyl methacrylate (GMA, (6)-2,3-epoxypropyl methylpropenoate, 95% by GC), N,N,N9,N9-tetramethylethylene diamine (TEMED), sodium perchlorate monohydrate and dimethyl sulfoxide (DMSO, ,0.01% water) were obtained from Fluka Chemie, AG, Buchs, Switzerland. L-lactide ((3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione, .99.5%) was purchased from Purac Biochem BV (Gorinchem, The Netherlands) and used without pretreatment. Stannous octoate (stannous (II) bis (2-ethylhexanoate), SnOct 2 , 95%) from Sigma Chemical Co., St. Louis, MO, USA was used as received. Toluene, 4-(N,Ndimethylamino) pyridine (DMAP, 99%) and N,N9-
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
carbonyl diimidazole (CDI, 98%) were purchased from Acros Chimica (Geel, Belgium). Tetrahydrofuran (THF) was distilled from LiAlH 4 immediately before use. Acetonitrile (HPLC, gradient grade) was obtained from Biosolve LTD (Valkenswaard, The Netherlands). Dialysis tubes (cellulose, molecular weight cut off (based on proteins)512–14 kDa) were purchased from Medicell International Ltd., London, UK. Potassium peroxodisulfate (KPS), sodium azide (NaN 3 ), perchloric acid (70%, p.a.) and the phosphate buffer components were obtained from Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) was purchased from OPG Farma, The Netherlands. Sodium- 125 iodide (Na 125 I) was from Amersham. Lysozyme (from chicken egg white) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bradford protein assay (Bio-Rad protein assay concentrate) and silver stain plus kit were obtained from Bio-Rad Laboratories (Veenendaal, The Netherlands). RPMI-1640 medium was purchased from Life Technologies (Breda, The Netherlands) and the cell proliferation kit II (sodium 39-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid; XTT) was used from Roche Diagnostics Nederland BV, Almere, The Netherlands.
2.2. Interleukin-2 Recombinant human interleukin-2 (Proleukin; 1.2 mg rhIL-2 per vial) was kindly provided by Chiron B.V. (Amsterdam, The Netherlands). rhIL-2 is a protein with a molecular weight of 15.3 kDa [23]. It is produced by recombinant DNA technology using an Escherichia coli strain, which contains a genetically engineered modification of the human IL-2 gene. This modified rhIL-2 differs from native human IL-2: the protein is not glycosylated, it has no N-terminal alanine and the serine at position 125 is substituted for cysteine. The biological activities of rhIL-2 and native human IL-2 are similar [23]. When the white lyophilized powder is reconstituted with 1.2 ml water each vial contains per ml solution: 1 mg (18310 6 IU) rhIL-2, 50 mg mannitol (5% w / v), and 0.2 mg (0.02% w / v) SDS, buffered with sodium phosphates to a pH of 7.5 (range 7.2–7.8). This solution is further described as ‘rhIL-2 stock solution’.
3
2.3. Methods 2.3.1. Radioiodination of rhIL-2 rhIL-2 was labeled with 125 I using the Iodo-gen method [24]. Briefly, a 50 ml aliquot (containing 25 mg) of Iodo-gen solution in dichloromethane was dispensed into a glass tube. The solvent was removed under a stream of nitrogen and 10 ml of 0.5 M Tris–HCl buffer (pH 7.4), 60 ml of 5% glucose containing 0.01% SDS, 15 ml of ‘rhIL-2 stock solution’ (1 mg / ml) and 8–10 ml of Na 125 I (.1 mCi) were added subsequently. After mixing, the mixture was incubated for 10 min at room temperature. The separation of labeled rhIL-2 from unreacted Na 125 I was performed by gel filtration using a Sephadex G-25 PD 10 column. As eluent 5% glucose containing 0.01% SDS was used. The labeling efficiency was more than 95% and the specific activity was 18 mCi / mg rhIL-2. The half-life of 125 I is 60 days [25]. 2.3.2. Stability of rhIL-2 in various buffers The stock solution of rhIL-2 (1 mg / ml) was diluted to 100 mg / ml with 0.9% NaCl; with 10 mM phosphate buffer (PB) or 10 mM phosphate buffered saline (PBS), 10 mM PB containing 0.01% SDS and 5% glucose, PB (10 mM and 100 mM) containing 0.1% SDS or 20 mM HEPES with or without 0.8% NaCl. The pH of the buffers was 7.2 and the total volume was 1 ml. Firstly, the stability of rhIL-2 in these buffers was tested for a short incubation period (15 min, room temperature). Secondly, the stability of rhIL-2 was studied over a longer time period (30 days) using the buffer (100 mM PB containing 0.1% SDS) in which rhIL-2 was recovered completely after 15 min of incubation. Now, 0.02% sodium azide (NaN 3 ) was added to avoid bacterial growth. The protein (36 mg / ml) was incubated in 15 ml buffer under physiological conditions (pH 7.2 and 378C) and samples were taken periodically. The samples were centrifuged (Eppendorf, 13 0003g) and 0.5 ml of the supernatant was removed and assayed for the presence of rhIL-2 by RP-HPLC (see Section 2.3.8). The same experiments were performed using 125 IrhIL-2. Briefly, 10 ml (0.9 mCi) of 125 I-rhIL-2 was added to rhIL-2 with a final concentration of 100
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mg / ml. The samples were centrifuged (Eppendorf, 13 0003g) and the number of counts in 0.5 ml of the supernatant and in the remaining resuspended fluid (0.5 ml) was determined using a gamma counter 1470 Wizard (EG&G Wallac, Turku, Finland).
2.3.3. Preparation of dextran hydrogels The polymers, dex-MA and dex-lactate-HEMA, were prepared from dextran T40 as previously described [26–28]. In brief: dex-MA was synthesized by the coupling of glycidyl methacrylate to dextran dissolved in DMSO using 4-(N,N-dimethylamino)pyridine as catalyst. For the synthesis of Dexlactate-HEMA, first L-lactide was grafted onto HEMA using stannous octoate as catalyst, yielding HEMA-lactate. After activation of the terminal hydroxyl group of HEMA-lactate with N,N9-carbonyldiimidazole, the resulting HEMA-lactate-CI was coupled to dextran in DMSO using 4-(N,N-dimethylamino)pyridine as catalyst to yield dex-lactateHEMA. Hydrogels were obtained by radical polymerization of dex-MA or dex-lactate-HEMA in aqueous solution according to the following general procedure [20]. For a hydrogel with an initial water content of 80% (w / w), 400 mg of methacrylated dextran was dissolved in 1460 ml phosphate buffer (PB; 100 mM, pH 7.2), in a 2-ml Eppendorf cup. To the dextran solution, 100 ml of KPS (20 mg / ml) in 100 mM phosphate buffer (PB, pH 7.2) was added and mixed well. Subsequently, 40 ml of a 20% (v / v) TEMED solution in PB (adjusted with 4 N HCl to pH 7) was added. After mixing, the resulting solution was allowed to polymerize for 1 h at room temperature. 2.3.4. Degradation of hydrogels After polymerization, the dex-lactate-HEMA hydrogels were removed from the cups, cut into a cylindrical shape (length approximately 2.5 cm, radius 0.45 cm), and accurately weighed (W0 ). The hydrogels were transferred into glass vials containing 15 ml of phosphate buffered saline (100 mM PB, 0.9% NaCl, 0.02% NaN 3 , pH 7.2) or phosphate buffer (100 mM PB) containing 0.1% SDS and 0.02% NaN 3 , pH 7.2. After incubation in a water bath at 378C, the weight of the hydrogels was determined at regular time points, and used to calculate the swelling ratio. The swelling ratio is
defined as the ratio of the weight of the hydrogel at time t (Wt ) and the initial weight of the hydrogel (W0 ) [20].
2.3.5. Release of proteins from dextran hydrogels The protein-loaded dex-MA and dex-lactateHEMA hydrogels were prepared according to the procedure described in Section 2.3.3. For lysozyme loaded gels, 25 ml of a 100 mg / ml lysozyme solution in PB was added to the polymer solution, yielding a loading of 1.25 mg lysozyme / g gel. For rhIL-2 loaded hydrogels, 420 ml of a 1.3 mg / ml rhIL-2 solution was added to the polymer solution, yielding a loading of 0.27 mg rhIL-2 / g gel. The release of lysozyme from the gels was determined after incubation of the protein loaded gel at 378C in vials containing 15 ml 100 mM PB containing 0.9% NaCl and 0.02% NaN 3 (PBS), pH 7.2. The rhIL-2 loaded gels were incubated in 15 ml of 100 mM PB containing 0.1% SDS and 0.02% NaN 3 , pH 7.2. The vials were gently shaken, and 1 ml samples were taken periodically and replaced by fresh buffer. The lysozyme concentration was measured with the Bio-Rad protein assay [29]. The rhIL-2 concentration was determined by RP–HPLC. 2.3.6. Release of 125 I-rhIL-2 from dextran hydrogels 125 I-rhIL-2 loaded hydrogels were prepared as described above. Before polymerization the polymer solution contained per ml 0.8 mCi 125 I-rhIL-2 and 0.27 mg non-labeled rhIL-2. After 1 h polymerization at room temperature, the gel was incubated in 15 ml of 100 mM PB10.1% SDS10.02% NaN 3 , pH 7.2. Periodically, 1 ml samples were taken and replaced by fresh buffer. The number of counts was measured with a gamma counter 1470 Wizard. The percentage of free label ( 125 I) in the release samples was determined by ITLCESG Instant Thin Layer Chromatography (silica gel impregnated glass fiber sheets; Gelman Sciences, Ann Arbor, MI, USA). As eluent 0.15 M sodium citrate pH 5.5 was used. The protein-loaded gels which were still present after 35 days, were transferred into a glass tube and the amount of remaining rhIL-2 in the gel was measured by using a gamma counter 1470 Wizard.
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
2.3.7. Adsorption of rhIL-2 onto dextran-based hydrogels To investigate the possible adsorption of rhIL-2 onto dextran-based hydrogels, gels with initial water content of 70% and DS 7-9 were incubated in an rhIL-2 solution with varying protein concentration. Solutions with 7 and 36 mg / ml rhIL-2 in 100 mM PB containing 0.1% SDS and 0.02% NaN 3 with or without 125 I-rhIL-2 (0.8 mCi) were used. Periodically, samples were taken and analyzed with RP– HPLC or a gamma-counter 1470 Wizard. 2.3.8. High-performance liquid chromatography rhIL-2 samples were analyzed by reversed phase– high-performance liquid chromatography (RP– HPLC) [30]. An LC Module I system (WatersE) ˚ with an analytical column (Jupiter, 5 mm C4 300 A, 15034.6 mm including a SecurityGuardE guard cartridge system with Widepore C4 (433 mm)) was used. All rhIL-2 samples were centrifuged for 5 min (13 0003g) and 100 ml of the supernatant was injected onto the column. A linear gradient was run from 40% A (water–acetonitrile 95:5; w / w; 100 mM sodium perchlorate (NaClO 4 ); 10 mM perchloric acid (HClO 4 )); 60% B (water–acetonitrile 5:95; w / w; 100 mM NaClO 4 ; 10 mM HClO 4 ) to 100% B in 10 min. The flow-rate was 1.0 ml / min and the column oven was set at 308C. UV detection at a wavelength of 205 nm was applied. Peak areas were determined with Millennium 2010 V. 2.15 software (Waters Associates Inc.). The total amount of methionine oxidized [31] and native rhIL-2 was calculated by using an rhIL-2 calibration curve over the range of 0.1–5 mg IL-2. The relative standard deviation for the intra-day precision was less than 3% and the inter-day standard deviation was 7%, which is within the range of the methodical error [32,33]. 2.3.9. Gel electrophoresis SDS–PAGE based on the method of Laemmli [34] was performed under denaturing (non-reducing and reducing) and native conditions. The Bio-Rad MiniProtean system was used with a 5% stacking gel and a 12.5% separating gel (thickness of 0.75 mm), both containing 0.4% SDS (denaturing conditions) or 0.1% SDS (native conditions). The rhIL-2 concen-
5
tration was in the range of 3–25 mg / ml. For denaturing conditions (non-reducing), sample buffer (60 mM Tris–HCl, pH 6.8; 25% (v / v) glycerol; 2% (v / v) SDS; 0.1% (w / v) bromophenol blue) was added to the samples in 1:4 (v / v) buffer to sample ratio and subsequently heated at 1008C for 5 min. For reducing conditions the sample buffer also contained b-mercaptoethanol (5% (v / v)). Native gel electrophoresis (non-reducing, non-boiled) was also performed. The sample buffer (60 mM Tris–HCl, pH 6.8; 25% (v / v) glycerol; 0.5% (v / v) SDS; 0.1% (w / v) bromophenol blue) was added to the samples in 1:4 (v / v) buffer to sample ratio. After loading the samples (24 ml sample per well), the gel was run for approximately 2 h at a voltage of 100 V with an electrophoresis buffer (0.3% (w / v) Tris; 1.4% (w / v) glycine; 0.1% (v / v) SDS). After running, the protein bands were visualized by silver nitrate staining. The marker contained lysozyme (Sigma, chicken egg white; Mw 15 kDa), ovalbumin (Sigma, chicken egg; Mw 40 kDa) and BSA monomer (ICN, Mw 66 kDa) in 100 mM PB containing 0.1% SDS. The concentration was 20 mg / ml for each protein. As control, rhIL-2 containing 6% of dimer was used (kindly provided by Chiron BV, Amsterdam, The Netherlands).
2.3.10. rhIL-2 bioassay The biological activity of rhIL-2 was measured by using an rhIL-2 dependent murine tumor-specific cytotoxic T cell line (CTLL-2) [35]. Samples of rhIL-2 released at different time points from dex-MA DS 9 and dex-lactate-HEMA DS 7 hydrogels with different initial water contents were tested. The rhIL2 concentration was determined by RP–HPLC. As control, rhIL-2 was incubated in the release buffer at 378C. Cells were cultured in RPMI-1640 medium supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin and 10% fetal calf serum in a humidified atmosphere of 5% CO 2 in air at 378C. The samples were diluted in steps of 1 in 10 in a range of 30 ng / ml to 0.03 ng / ml in a 96-wells plate (100 ml per well; in triplicate). To each well 50 ml of CTLL-2 cell suspension (10 000 cells) was added and the plate was incubated for 2 days in a humidified atmosphere of 5% CO 2 in air at 378C. Then, 50 ml of XTT solution was added to each well
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and after 4–12 h the proliferation was measured by using a Cell Proliferation Kit II.
3. Results and discussion
3.1. Stability of rhIL-2 As pointed out in the introduction, the rhIL-2 formulation upon reconstitution and dilution is critical since the protein has limited aqueous solubility [3,5,24]. To examine in which aqueous solution rhIL-2 is stable, the protein was incubated in various media. The recovery of rhIL-2 after 15 min incubation at room temperature was less than 80% when the protein was diluted in 0.9% saline, PB with or without saline, PB containing 0.01% SDS and 5% glucose, or in HEPES buffer with or without saline. The addition of a detergent, 0.1% SDS, to the PB without saline resulted in complete solubilization of rhIL-2 (protein recovery .95%). rhIL-2 also showed a good long term stability in this buffer solution: in a time period of 30 days only a slight decrease of approximately 10% in rhIL-2 recovery was detected. Importantly, rhIL-2 incubated in this buffer showed no detectable loss in biological activity (14 days, 378C). Moreover, in line with this observation aggregates of rhIL-2 were not detected as determined with native gel electrophoresis (Fig. 1A; lane 9). Therefore, the release studies of rhIL-2 from hydrogels were performed in 100 mM PB containing 0.1% SDS and 0.02% NaN 3 , pH 7.2.
3.2. Degradation of dex-lactate-HEMA hydrogels From the stability study of rhIL-2 in different aqueous media, it was shown that rhIL-2 was stable in 100 mM PB containing 0.1% SDS and not in PBS. Since the degradation of dextran-based gels was only examined in PBS [20], these gels were incubated in PB containing 0.1% SDS to investigate whether SDS has an effect on the degradation process. Fig. 2 shows a representative example of the swelling behavior of a dex-lactate-HEMA gel with initial water content of 80% and DS of 7 in PBS and in PB containing 0.1% SDS. The hydrogel showed in both buffers a progressive swelling, which is caused by hydrolysis of the lactate and / or carbonate esters
Fig. 1. Native gel electrophoresis (A) and SDS–PAGE analysis under reducing conditions (B) of rhIL-2 release samples of dexMA DS 9 and dex-lactate-HEMA DS 7 gels both with initial water content of 90%. Lane 1: Molecular weight marker (BSA, ovalbumin and lysozyme); lane 2: rhIL-2 standard containing 6% dimer; lanes 3–5: rhIL-2 released from dex-MA gel incubated in 100 mM PB10.1% SDS after 1, 3 and 7 days, respectively; lanes 6–8: rhIL-2 release from dex-lactate-HEMA gel incubated in 100 mM PB10.1% SDS after 1, 3 and 7 days, respectively. As control, rhIL-2 was incubated for 17 days in 100 mM PB containing 0.1% SDS at 378C (1A; lane 9).
present in the crosslinks, followed by a dissolution phase [20]. The time in which the gel is completely degraded is defined as the degradation time of the gel. The degradation time of the dex-lactate-HEMA hydrogel was longer when the buffer contained SDS (Fig. 2 and Table 1). This can be ascribed to the interaction of SDS with the hydrophobic lactate units present in the hydrogel. A likely explanation is that shielding diminishes the accessibility of water molecules to hydrolyze the lactate ester bonds, resulting in a slower degradation of the dex-lactate-HEMA
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
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Fig. 2. Swelling behavior of dex-lactate-HEMA hydrogels with DS 7 and an initial water content of 80% in 100 mM PBS (h) or in 100 mM PB containing 0.1% SDS, pH 7.2 (j), at 378C. The values are the average of two independent measurements.
hydrogel and an increased degradation time in an SDS-containing buffer.
Fig. 3. Representative HPLC chromatogram of rhIL-2 standard (A), rhIL-2 released from an 80% dex-MA DS 9 gel after 5 days (B) and rhIL-2 released from a 90% dex-lactate-HEMA DS 7 gel after 5 days (C; peaks with retention times of 4–6 min correspond to degradation products of the dex-lactate-HEMA gel).
3.3. Protein release from dextran-based hydrogels The release of rhIL-2 and lysozyme from nondegradable dex-MA hydrogels and from degradable dex-lactate-HEMA gels with different crosslink densities was studied. In contrast to the rhIL-2 standard (Fig. 3A), HPLC chromatograms of rhIL-2 released from the hydrogels showed two peaks with a retention time of 8 and 9.5 min (Fig. 3B,C). The peak with the largest retention time (9.5 min) contains native as well as methionine oxidized forms (20– 33%) of rhIL-2. The peak with the shortest retention time (8 min) contains rhIL-2 in which all methionine Table 1 The degradation time of dex-lactate-HEMA DS 7 hydrogels with varying initial water content (w / w) in 100 mM PBS, pH 7.2 and 100 mM PB containing 0.1% SDS, pH 7.2 Initial water content (%) 90 80 70 60
Degradation time (days) PBS
PB10.1% SDS
5 9 18 23
7 15 31 36
residues at position 103 are oxidized and also oxidized methionine residues at the other positions [31,36]. Fig. 4 shows the release of rhIL-2 (A) and lysozyme (B) from dex-MA hydrogels with varying initial water content. As demonstrated previously [22], gels with a DS around 10 are dimensionally stable (do not show additional water uptake once incubated in an aqueous solution). This means that the equilibrium water content is the same as the water content of the gels after preparation. Fig. 4 shows that, as expected, with decreasing water content of the hydrogel, the release rate as well as the total release of both proteins decrease. Similar release profiles of rhIL-2 from dex-MA hydrogels were obtained when 125 I labeled rhIL-2 was used (not shown). Both HPLC and radioactivity measurements showed that rhIL-2 was not quantitatively released from the investigated gels in the time frame studied. The non-released part was still entrapped in the hydrogel: using 125 I-rhIL-2, it was shown that 6 and 80% of the initial amount of protein was still present after 35 days in gels with initial water
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Fig. 4. Release of rhIL-2 (A) and lysozyme (B) from dex-MA hydrogels with DS 9 and an initial water content of 90% (j), 80% (♦), 75% (m), 70% (d), 60% (.) and 50% (w) in 100 mM PB pH 7.2, with (rhIL-2) or without 0.1% SDS, at 378C. Duplicate release curves varied less than 10% as indicated by the error bars. Inserts show the initial proteins release of rhIL-2 from these hydrogels versus the square root of time.
contents of 90 and 70%, respectively. The total amount of released rhIL-2 and still entrapped rhIL-2 in the gels resulted in a recovery of more than 90%. As shown in Fig. 4, substantial amounts of rhIL-2 and lysozyme were released from dex-MA hydrogels
with initial water content of 75–90% and 50–90%, respectively. The initial release of both proteins from these hydrogels is proportional to the square root of time as depicted in the insert of Fig. 4A and B. This indicates that the pore sizes of the hydrogels are
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
larger than the hydrodynamic radius of the protein [22] and that the release followed Fickian diffusion [37,38]. Fig. 4 shows that for rhIL-2 a slower release was observed than for lysozyme from gels with the same network characteristics. Moreover, rhIL-2 was marginally released (less than 10% in 25 days) from dex-MA gels with an initial water content of 70% or lower (Fig. 4A), whereas a significant amount of lysozyme was released from gels with the same composition (Fig. 4B). This suggests that in gels with an initial water content of #70% rhIL-2 is almost fully entrapped in the meshes of the gels, whereas (part of) lysozyme is still able to diffuse through the network. The entrapment of rhIL-2 is unexpected, since the molecular weight of the monomer of rhIL-2 is almost the same as that of lysozyme. Possible explanations might be interaction of rhIL-2 with the hydrogel matrix, formation of aggregates or interference by SDS. Native gel electrophoresis showed that some dimers of rhIL-2 are released from a dex-MA gel with initial water content of 90% (Fig. 1A lanes 3–5). To investigate whether these dimers consist of disulfide-linked protein species, SDS–PAGE under non-reducing and reducing conditions was performed. As shown in Fig. 1B (lane 3–5), the dimers were still present under reducing conditions suggesting that the dimers are linked by covalent bonds other than disulfide bridges. However, the amount of rhIL-2 dimer released is approximately 5–10% based on rhIL standard containing 6% dimer (Fig. 1B; lane 2). Thus, the entrapment of rhIL-2 in dex-MA gels with initial water content of 70% or lower can not be fully explained by the formation of dimers. Possible adsorption of rhIL-2 to the hydrogel matrix was investigated by incubation of a dex-MA gel in an rhIL-2 solution. This resulted in no adsorption of rhIL-2 (RP–HPLC, 125 I-rhIL-2) to the gel, indicating that the protein has no interaction with the dex-MA hydrogel matrix. Since it is likely that SDS interacts with the crosslinks (polymerized methacrylate groups) in the gel, the matrix becomes negatively charged. On the other hand, SDS will also interact with rhIL-2, resulting in a larger hydrodynamic protein diameter and an overall negative charge. It can be expected that the mobility of the negatively charged SDS coated rhIL-2 molecules in such a
9
matrix is restricted and might explain the observed entrapment of rhIL-2 in matrices with small pores [39]. The release of both rhIL-2 and lysozyme from degradable dex-lactate-HEMA and dex-HEMA hydrogels was also investigated. Fig. 5A and B show the release profiles of rhIL-2 and lysozyme, respectively, from dex-lactate-HEMA gels with varying initial water content. Since the degradation products
Fig. 5. (A) Release of rhIL-2 from dex-lactate-HEMA hydrogels with DS 7 and an initial water content of 90% (j), 80% (♦), 75% (m), 70% (d) and 60% (.) in 100 mM PB containing 0.1% SDS, pH 7.2, 378C. (B) Release of lysozyme release from dex-lactateHEMA hydrogels with DS 7 and an initial water content of 90% (j), 80% (♦), 70% (d) and 60% (.) in 100 mM PBS, pH 7.2, 378C. Insert shows the initial release of lysozyme from these hydrogels versus the square root of time.
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(among others oligomethacrylates) interfered with the protein assay [20] and most likely with the HPLC analysis as well, the release of both proteins from the degrading hydrogels was quantified until the matrix of the gels started to dissolve. As expected, by decreasing the initial water content of the gel, the protein release rate decreases. As observed for the non-degradable dex-MA gels, the release of lysozyme from the dex-lactate-HEMA gels showed a square root of time dependency (insert of Fig. 5B). This indicates that the pore sizes of these hydrogels are larger than the hydrodynamic radius of lysozyme and the lysozyme release from these hydrogels therefore followed Fickian diffusion. The release of rhIL-2 from a highly hydrated dex-lactate-HEMA gel (initial water content of 90%) showed a square root of time dependency as well (data not shown). In contrast to the marginal rhIL-2 release from nondegradable dex-MA gels with initial water content of 70% or lower (Fig. 4A), rhIL-2 was released from degradable dex-lactate-HEMA gels with similar crosslink densities (Fig. 5A). This can be ascribed to hydrolysis of the lactate esters present in the crosslinks, resulting in an increased mesh size of the gel in time. Now, the protein release is controlled by the swelling of the matrix [20]. An almost zero-order release of rhIL-2 in a time period of approximately 5–15 days was obtained from the dex-lactate-HEMA gels (initial water content ,90%). The observed zero-order release of rhIL-2 can be explained as described previously for the release of immunoglobulin G from gels with the same composition [20]. On the one hand the diffusion coefficient of the protein increases in time due to swelling of the matrix; on the other hand the release rate decreases due to increase in hydrogel size and decrease in concentration gradient. Obviously in the dex-lactate-HEMA hydrogels, these factors compensate each other, resulting in an almost zero-order release. To investigate the release of rhIL-2 from dexlactate-HEMA hydrogels over a longer time period, 125 I labeled rhIL-2 was encapsulated in these hydrogels. The release profiles established by measuring the radioactivity of the samples matched with those determined by RP–HPLC, especially for a hydrogel with a high initial water content (Fig. 6). Moreover, the recovery of 125 I-rhIL-2 after complete degra-
Fig. 6. Release of rhIL-2 loaded dex-lactate-HEMA hydrogels as measured by radioactivity of co-incorporated 125 I-rhIL-2 (closed symbols) or RP–HPLC (open symbols). The initial water content of the hydrogels was 90% (squares) and 70% (circles).
dation of the hydrogels was approximately 90% as shown in Fig. 6. As was true for the dex-MA hydrogels, rhIL-2 released from the dex-lactateHEMA gels consisted of mainly monomer of rhIL-2 and some covalently bonded dimers (5–10%; Fig. 1A1B lanes 6–8). The release of rhIL-2 from degradable dex-HEMA gels with fixed initial water content of 70% and different degree of substitutions, DS 8 and 14, was also studied (Fig. 7). 125 I-rhIL-2 was used since the degradation products of dex-HEMA hydrogel might interfere with HPLC analysis. The release of rhIL-2 is slower from a degradable dex-HEMA gel as compared with a dex-lactate-HEMA gel with comparable network characteristics (DS and initial water content; Fig. 7). These differences in release profile can be explained by the faster degradation of the dex-lactate-HEMA gel compared to dex-HEMA gels (1 and 3 months, respectively). Furthermore, by increasing the degree of substitution of the dex-HEMA gels, the rhIL-2 release rate decreases. It is obvious that increasing the DS at a fixed water content results in a network with a higher crosslink density. To dissolve the network more crosslinks have to be hydrolyzed resulting in a slower swelling, which is associated with a slower protein release.
´ et al. / Journal of Controlled Release 78 (2002) 1 – 13 J. A. Cadee
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polyphosphate pastes showed a loss of approximately 70% in biological activity [40]. Gel electrophoresis showed that rhIL-2 was mainly released as monomer from both non-degradable dex-MA and degradable dex-lactate-HEMA gels (Fig. 1A and B). Since the concentration of released rhIL-2 was too low for spectroscopic analysis like circular dichroism or fluorescence spectroscopy, the protein integrity was studied by measuring its biological activity. As shown in Table 2, rhIL-2 released from hydrogels has 50–70% of its original activity as evaluated by the CTLL-2 proliferation assay. The nature of the observed loss of the bioactivity is under present investigation. Fig. 7. Release of 125 I-rhIL-2 from dex-HEMA hydrogels with DS 8 (♦) and DS 14 (d) with initial water contents of 70%. As control, the release of rhIL-2 from a 70% dex-lactate-HEMA DS 7 (j) gel.
3.4. Biological activity Proteins that are encapsulated in release systems, can be damaged during the formulation, storage and release, which can result in loss of biological activity [16]. Egilmez et al. reported that half of the biological activity of rhIL-2 is lost during the encapsulation process of the protein in polylactic acid microspheres. This is most likely a result of protein denaturation, caused by the presence of organic solvents [13]. RhIL-2 released from biodegradable
4. Conclusions In this paper, we demonstrate that the release of rhIL-2 from dextran-based hydrogels can be tailored by the crosslink density and the type of ester group present in the crosslink. From non-degradable dexMA gels with an initial water content above 70% the rhIL-2 release was governed by diffusion, whereas in gels with an initial water content of 70% or lower the protein was entrapped. The rhIL-2 release from degradable dex-lactate-HEMA gels was determined by diffusion and / or hydrogel degradation. Importantly, the degradable rhIL-2 loaded hydrogels showed an almost zero-order release in a time period of approximately 5–15 days. rhIL-2 released from the
Table 2 Biological activity of rhIL-2 released from dex-MA and dex-lactate-HEMA gels with initial water content (w / w) of 70% and 90% at different time points Formulation
Water content (%)
Day
Biological activity (%)a
Dex-MA
90
Dex-lactate-HEMA
90
2 8 2 7 11
79 65 65 81 64
Dex-MA
70
Dex-lactate-HEMA
70
1 19 1 24
46 53 57 55
a
The biological activity is related to freshly reconstituted rhIL-2 in PB containing 0.1% SDS. The values are the mean of two measurements that deviated 5–10%.
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dextran hydrogels investigated was mainly in its monomeric form with a partial preservation of the biological activity (50–70%). rhIL-2 loaded dextranbased hydrogels are promising protein delivery systems for tumor immunotherapy.
[10]
[11]
Acknowledgements [12]
This investigation was supported by the Dutch Technology Foundation, research grant UFA 55.3931.
[13]
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